Brendan C.

Edit 73 …
Week of 12/1/13-12/7/13




FPLC.PNG
Figure: FPLC spec results. Tubes collected = 39-48 for reconcentration.


SpecPostFPLC.jpg
Figure: PreFPLC spec results after Ni-NTA assay of new batch of protein NDM-1.
testassay.png

Figure 1: Verification assay of protein activity after new batch of NDM-1 made.

Pictureapples.png
Figure: Image of characterization of protein solutions at different stages of purification.


Analysis: We now have enough protein to do 4,500 spec analyses for assays. We've shown that the protein solution is more pure and that the activity is
present. FPLC results were quite good and initial protein concentration post Ni-NTA column was very high. The gel indicates very good purity of protein in the FPLC lane. Note that the sample from FPLC was diluted prior to adding to the gel due to the high concentration of protein.

Week of 11/24-11/30/13

preFPLC.png
Figure: PreFPLC spec results after Ni-NTA assay of new batch of protein NDM-1.


Very high concentration of protein. Not sure if all of it is the target protein however. Gel characterization indicates a lot of contaminating protein
so FPLC will be used to get rid of it.
Week of 11/17/13-11/23/13


11/23/13: Final attempt at activity assay. - @ 490nm
Activity Assay:
0.25ug in 10uL buffer (so add 19uL buffer per 1uL solution)
Dilute to 20uM nitrocefin in 0.5mL soln in cuvette
50mM Hepes, 10uM ZnSO4. <Add until Nitrocefin+Buffer=490uL>

Inhibition Assay:




11/19/13 - HF9 plates GOLD results are in.

Table 1: GOLD docking results of 4EY2 vs HF9 plates (~40,000 ligands). Top 30 scores shown.
#
 Score
 S(PLP)
 S(hbond)
 S(cho)
 S(metal)
 DE(clash)
 DE(tors)
 intcor
 time
 name
1
 102.49
 -77.9
 1.88
 0
 3.96
 0
 2.62
 0.41
 44.488
 'KM10420SC'
2
 100.93
 -89.68
 0.6
 0
 2
 0
 2.55
 2.55
 139.309
 'SPB06607SC'
3
 100.64
 -65.71
 2.79
 0
 4.76
 0
 1.86
 1.75
 141.639
 'CD09635SC'
4
 99.31
 -91.4
 0.65
 1
 1
 0.31
 3.3
 3.26
 192.226
 'HTS11951SC'
5
 98.36
 -75.17
 4.69
 0
 2.1
 0
 2.23
 0.99
 66.862
 'NRB05197SC'
6
 97.81
 -74.75
 2.65
 0
 2.87
 0
 1.86
 1.63
 196.913
 'BTB13323SC'
7
 97.65
 -84.3
 0
 1
 1.88
 0
 1.79
 2.65
 21.541
 'HTS08270SC'
8
 97.54
 -81.81
 1
 0
 2.63
 0
 2.16
 0.99
 28.486
 'HTS09917SC'
9
 97.32
 -81.04
 1
 0
 2.94
 0
 3.46
 2.58
 176.638
 'HTS02114SC'
10
 96.63
 -79.59
 0.88
 0
 2.97
 0.18
 3.18
 3.01
 92.801
 'RH01689SC'
11
 96.52
 -76.77
 0
 0
 3.94
 0
 2.08
 0.13
 28.004
 'SCR00160SC'
12
 96.27
 -99.1
 0.43
 0
 0
 0
 2.16
 0.2
 87.649
 'HTS07745SC'
13
 95.73
 -80.39
 0.8
 0
 2.86
 0
 3.22
 2.19
 116.015
 'HTS08667SC'
14
 95.71
 -80.05
 2
 0
 1.99
 0
 2.26
 2.22
 142.861
 'NRB01104SC'
15
 95.53
 -80.37
 0.9
 0
 2.92
 0.58
 2.3
 0.13
 85.736
 'HTS08664SC'
16
 95.35
 -67.09
 2.93
 0
 3.89
 0
 2.56
 1.14
 116.92
 'HTS08000SC'
17
 94.86
 -72.23
 1.99
 0
 3
 0
 0.73
 0.12
 115.001
 'HTS08318SC'
18
 94.85
 -73.47
 2.71
 0
 2.94
 0
 4.44
 4.53
 141.635
 'HTS02720SC'
19
 94.84
 -68.78
 1.38
 1
 3.5
 0.48
 1
 0.38
 36.759
 'HTS01400SC'
20
 94.84
 -86.28
 0
 0
 2
 0
 1.98
 0.52
 60.642
 'CD08739SC'
21
 94.78
 -80.72
 0
 0
 2.83
 0
 1.54
 0.13
 111.982
 'CD12049SC'
22
 94.76
 -78
 0.8
 0
 2.66
 0
 1.78
 1.95
 18.199
 'HTS12773SC'
23
 94.18
 -73.09
 0
 0
 3.99
 0
 1.55
 0.18
 24.803
 'HTS01661SC'
24
 94.17
 -77.29
 0
 0
 3.44
 0.15
 3
 2.41
 21.914
 'NRB04246SC'
25
 93.95
 -81.61
 0.99
 0
 2
 0
 1.85
 1.09
 64.839
 'SPB03389SC'
26
 93.84
 -77.69
 0
 0
 2.94
 0
 1.86
 2.25
 30.367
 'SEW05237SC'
27
 93.68
 -73.59
 3.63
 0
 1.97
 0
 2.46
 2.3
 129.884
 'HTS11140SC'
28
 93.65
 -79.83
 0.5
 0
 2.65
 0
 2.98
 2.25
 113.86
 'SPB01505SC'
29
 93.38
 -76.64
 0
 0
 3.67
 0
 2.76
 0.2
 22.886
 'HTS00696SC'
30
 93.35
 -71.81
 1.32
 1
 2.96
 0
 2.71
 2.26
 129.794
 'HTS05874SC'
Week of 11/10/13-11/16/13

11/15/13: Protein Purification and Characterization
external image 5kfzo.jpg
Figure 1: FPLC results of purification of second batch of NDM-1.

In short, the FPLC seems to have worked. We took sample tubes 41-46 and reconcentrated to 1mL. The resulting solution was nanodropped
and determined to be 0.58mg/mL. This was conducted on a batch of protein on which expression was begun earlier in the week. After FPLC
and concentration was completed, snapfreezing was conducted.

Meanwhile, another batch of protein expression is occurring. To this batch, Ni-NTA column purification was conducted.

external image 5kfQD.png
external image 5kfRO.png
These results show significantly less protein in the elution 2 after changing wash imidazole concentration to 15mM.



11/12/13: Protein purification
external image 5hDe1
Figure 1: Nanodrop of elution 1 after Ni-NTA purification.

Analysis: No distinct 280 peak suggests a not-so-great protein expression. The high 230 peak suggests
considerable contamination. Some things to consider for the future = FPLC and rerunning the Ni-NTA column.
0.56mg/mL is not a terrible concentration however, though repurification will most likely be coming up.

external image 5kfYW.jpg

Figure 3: Batch 1 of protein made after Ni-NTA purification. Lane 1 indicates Elution 1 and lane 2 indicates Elution 2.
Indicates reason to use FPLC to purify batch 1.

The gel for batch 2 is currently destaining after Ni-NTA purification.

11/11/13: Protein expression continuing.

Table 1: OD600 to 0.5 abs data prior to IPTG induction (set 2)
Time
 mL added of small culture
 Large culture 1 OD600
 Large culture 2 OD600
2:14
 0
 0

5
 0

10
 0.021

15
 0.08

18
 0.1
 0.16
3:18
0.12
 0.2
3:48
0.29
 0.478
3:58
0.438
 0.544
4:13
0.567





Week of 11/3/13-11/9/13

11/8/13: 4EY2 vs HF9plates screening still incomplete on the DDFE.
Protein expression continuing.

Table 1: OD600 to 0.5 absorbance data prior to IPTG induction.

Flask
 Time
 OD(600)
 Flask 2
 Time
 OD600
1
 2:09
 0.099
 2
 2:14
 0.13






Added to incubator





3:05
 0.087
3:07PM
 0.08

330
 0.101
330
 0.1

412
 0.181
412
 0.209

4:52
 0.369
4:46
 0.422

5:04
 0.452
4:56
 0.45

5:10
 0.503
5:05
 0.51

11/6/13: 4EY2 vs HF9plates screening begun.
-Protein expression begun - small scale culture

11/5/13: Screened 4EY2 vs CBDiversity in GOLD.
Plates of BL21(DE3) transformed with pet27b+NDM1-betalactamase made.

Table 1: 4EY2 docked to CBDiversity in GOLD (top 30 shown).

#
 Score
 S(PLP)
 S(hbond)
 S(cho)
 S(metal)
 DE(clash)
 DE(tors)
 intcor
 time
 name
1
 109.78
 -50.03
 0.4
 0
 9.89
 0
 0.57
 0.32
 18.27
 '5587264'
2
 109.44
 -62.12
 3.27
 0
 6.73
 0
 1.56
 0.25
 85.953
 '7784870'
3
 108.17
 -81.24
 0.02
 0
 4.9
 0
 1.82
 1.12
 95.191
 '7638084'
4
 107.91
 -85.25
 1
 0
 3.77
 0
 1.79
 0.44
 112.468
 '5321217'
5
 107.4
 -40.78
 3
 0
 9.9
 0
 2.26
 2.32
 16.458
 '5238378'
6
 107.2
 -67.07
 1.92
 0
 5.85
 0
 0.78
 0.62
 97.993
 '7565278'
7
 107.18
 -70.79
 0.78
 0
 5.96
 0.01
 1.29
 0.72
 93.248
 '7587987'
8
 106.84
 -70.95
 1.92
 0
 5.7
 0
 3.29
 2.45
 94.92
 '7746383'
9
 106.48
 -65.93
 2.87
 0
 5.95
 0
 2.36
 0.92
 76.339
 '6639676'
10
 106.36
 -66.66
 2.42
 0
 5.93
 0
 1.89
 0.48
 37.107
 '5378944'
11
 106.24
 -87.06
 0
 0
 3.88
 0.41
 3.97
 3.57
 133.13
 '6462982'
12
 106.12
 -56.24
 3
 0
 7.67
 0
 2.75
 0.34
 65.994
 '5157370'
13
 106.07
 -81.31
 1
 0
 3.98
 0
 1.14
 0.17
 98.259
 '7009439'
14
 105.8
 -67.86
 2.15
 0
 5.88
 0.08
 1.99
 0.14
 118.286
 '5378829'
15
 105.53
 -74.01
 0
 1
 5.73
 0.38
 3.29
 0.92
 144.458
 '6457101'
16
 105.5
 -65.38
 1.94
 0
 5.85
 0
 0.74
 0.57
 55.16
 '7584104'
17
 105.46
 -61.13
 0
 0
 7.87
 0
 2.54
 2.18
 24.301
 '9048987'
18
 105.36
 -64.75
 2.54
 0
 5.91
 0
 1.52
 0.58
 33.022
 '5378880'
19
 105.31
 -59.56
 0
 0
 7.99
 2.64
 1.63
 3.73
 29.417
 '5538434'
20
 105.28
 -74.85
 0.17
 0
 5.59
 2.41
 2.77
 4.33
 138.844
 '7722178'
21
 105.16
 -65.55
 1.67
 0
 5.97
 0
 0.67
 0.11
 29.696
 '7644832'
22
 105.12
 -60.2
 0
 0
 7.73
 0
 0.76
 0.06
 52.8
 '6052424'
23
 105.1
 -59.88
 0
 0
 7.85
 0
 0.98
 0.06
 34.304
 '6052410'
24
 104.94
 -64.67
 2
 0
 5.88
 10.48
 0.96
 11.38
 147.529
 '7551148'
25
 104.76
 -59.9
 0
 0
 7.82
 0
 1.05
 0.06
 51.932
 '6052398'
26
 104.7
 -76.95
 2.08
 0
 3.96
 1.21
 1.82
 2.4
 98.638
 '7589134'
27
 104.36
 -77
 0
 0
 5.61
 0
 3.27
 0.23
 80.345
 '7583099'
28
 104.33
 -81.93
 0
 1
 3.88
 0
 3.32
 2.77
 39.927
 '7632139'
29
 104.28
 -72.6
 0.97
 0
 4.88
 0
 1.06
 1.6
 77.234
 '6422125'
30
 104.22
 -58.16
 0
 0
 7.91
 0.9
 2.26
 3.68
 22.215
 '5226803'




Week of 10/27/13-11/2/13

10/31/13: 4EY2 Docking to CB306 in GOLD

Table 1: 4EY2 docked to CB306 in GOLD.
#
 Score
 S(PLP)
 S(hbond)
 S(cho)
 S(metal)
 DE(clash)
 DE(tors)
 intcor
 time
 name
1
 44.12
 -39.11
 2
 0
 0
 0
 1.7
 2.4
 4.123
 '5739880'
2
 41.77
 -22.9
 6.62
 0
 0
 0
 0.96
 0.33
 4.425
 '7726180'
3
 40.48
 -36.14
 1.12
 0.81
 0
 0
 0.8
 0.14
 4.472
 '9027311'
4
 40.33
 -28
 5
 0
 0
 0
 1.42
 0.16
 4.122
 '7575772'
5
 40.21
 -35.49
 1.67
 0
 0
 0
 0.4
 0.41
 4.142
 '7951649'
6
 39.89
 -32.68
 2.91
 0
 0
 0
 0.81
 0.09
 3.261
 '5978818'
7
 39.53
 -39.77
 0
 0
 0
 0
 0.61
 0.97
 4.059
 '5627579'
8
 39.38
 -31.46
 3
 0
 0
 0
 0.55
 0.01
 1.761
 '9040855'
9
 38.86
 -34.19
 2.63
 0
 0
 0
 1.79
 0.11
 3.507
 '7543758'
10
 38.83
 -31.2
 2.78
 0
 0
 0
 0.37
 0.01
 2.827
 '5761107'
11
 38.73
 -34.5
 2
 0
 0
 0.01
 0.98
 0.15
 5.063
 '7746693'
12
 38.71
 -33.31
 2.34
 0
 0
 0
 2.33
 3.04
 4.02
 '7623092'
13
 38.63
 -32.39
 3.13
 0
 0
 0
 1.63
 0.07
 3.724
 '7814849'
14
 38.53
 -37.81
 1.36
 0
 0
 0
 1.79
 0.23
 4.078
 '7938694'
15
 38.13
 -27.15
 4
 0.83
 0
 0
 3.05
 2.61
 0.958
 '6629394'
16
 38.12
 -34.02
 1.66
 0
 0
 0
 0.89
 0.54
 5.123
 '5654317'
17
 38.11
 -33.11
 1.8
 0
 0
 0
 0.21
 0.02
 2.869
 '6664007'
18
 38.04
 -31.28
 2.78
 0
 0
 0
 0.81
 0.01
 3.924
 '5957448'
19
 37.93
 -30.82
 2.93
 0
 0
 0
 0.84
 0
 0.964
 '9061037'
20
 37.83
 -31.33
 2.6
 0
 0
 0
 1.34
 0.86
 4.983
 '7558664'
21
 37.7
 -32.81
 1.98
 0
 0
 0
 0.91
 0.78
 4.577
 '5525637'
22
 37.7
 -36.33
 0.74
 0
 0
 0
 0.43
 0.02
 4.64
 '5728150'
23
 37.62
 -21.09
 6
 0
 0
 0
 0.91
 0.24
 3.036
 '5467784'
24
 37.57
 -21.33
 6.24
 0
 0
 0
 1.44
 0.1
 3.472
 '5192853'
25
 37.46
 -33.34
 1.81
 0
 0
 0
 0.95
 0.52
 3.41
 '5241283'
26
 37.26
 -36.94
 0.98
 0
 0
 0
 1.37
 0.12
 2.984
 '7766330'
27
 37.25
 -32.89
 2
 0
 0
 0
 0.86
 0.08
 3.654
 '7724431'
28
 37.19
 -32.08
 2.18
 0
 0
 0
 0.85
 0.28
 3.42
 '7698680'
29
 36.98
 -27.47
 3.76
 0
 0
 0
 1.95
 2.09
 3.615
 '7676009'
30
 36.96
 -36.31
 1
 0
 0
 0
 1.32
 0.28
 4.869
 '7930734'
31
 36.86
 -33.22
 1.99
 0
 0
 0
 1.19
 0.03
 2.73
 '5531413'
32
 36.79
 -29.28
 2.99
 0
 0
 0
 0.74
 0.02
 3.6
 '7935514'
33
 36.58
 -23.03
 4.92
 0
 0
 0
 0.69
 0.1
 3.613
 '5282931'
34
 36.56
 -30.22
 2.98
 0.35
 0
 0
 1.83
 0.01
 2.29
 '9045960'
35
 36.38
 -32.31
 1.57
 0
 0
 0
 0.33
 0.02
 3.36
 '5158497'
36
 35.76
 -36.72
 0.06
 0
 0
 0
 1.23
 1.32
 2.673
 '9038774'
37
 35.75
 -28.78
 2.74
 0
 0
 0
 0.63
 0.01
 1.985
 '7575112'
38
 35.65
 -28.48
 2.61
 0
 0
 0
 0.35
 0.05
 3.216
 '7741517'
39
 35.64
 -34.55
 1.58
 0
 0
 0
 1.84
 0.01
 2.2
 '7948268'
40
 35.62
 -32.98
 1.38
 0
 0
 0
 0.77
 0.03
 3.843
 '5193892'
41
 35.58
 -31.43
 1.55
 0
 0
 0
 0.25
 0.03
 2.64
 '7437091'
42
 35.52
 -31.42
 1.96
 0
 0
 0
 0.92
 0.04
 2.414
 '7608656'
43
 35.39
 -30.63
 1.96
 0
 0
 0
 0.62
 0.07
 4.068
 '9062573'
44
 35.31
 -34.26
 0.99
 0
 0
 0
 1.71
 1.49
 3.268
 '7911350'
45
 35.17
 -31.54
 2
 0
 0
 0.01
 1.94
 1.52
 2.783
 '5301198'
46
 35.1
 -19.45
 5.68
 0
 0
 0
 0.69
 0.02
 2.688
 '6433056'
47
 35.09
 -34.17
 0.51
 0
 0
 0
 0.32
 0.03
 2.53
 '7870889'
48
 35.06
 -32.57
 1.54
 0
 0
 0
 1.42
 0.72
 6.202
 '6956655'
49
 34.97
 -29.09
 2
 0
 0
 0
 0.07
 0.02
 2.739
 '7829885'
50
 34.96
 -30.63
 1.59
 0
 0
 0
 0.21
 0
 2.61
 '5215541'
51
 34.79
 -28.9
 2
 0
 0
 0
 0.06
 0.01
 2.343
 '9056306'
52
 34.78
 -18.82
 6
 0
 0
 0
 1.09
 0.08
 2.968
 '7842136'
53
 34.58
 -29.5
 2.62
 0
 0
 0
 1.42
 0.07
 3.525
 '5921021'
54
 34.51
 -28.57
 2
 0
 0
 0
 0.12
 0.19
 1.081
 '7115698'
55
 34.47
 -30.63
 2.21
 0
 0
 0
 1.57
 0.18
 4.038
 '7957875'
56
 34.37
 -31.98
 0.99
 0
 0
 0
 0.34
 0.05
 3.553
 '7493042'
57
 34.29
 -26.39
 3.01
 0
 0
 0
 0.58
 0.02
 1.476
 '5135469'
58
 34.04
 -32.52
 1.04
 0
 0
 0
 0.81
 0.02
 3.1
 '7963108'
59
 34.02
 -28.8
 1.95
 0
 0
 0
 0.34
 0.02
 2.642
 '7684808'
60
 34
 -31.76
 1.56
 0
 0
 0.15
 2.15
 2
 4.592
 '7965868'
61
 33.95
 -26.18
 2.97
 0
 0
 0
 0.57
 0
 3.255
 '5115021'
62
 33.89
 -29.91
 1.61
 0
 0
 0
 0.46
 0.06
 3.132
 '7723709'
63
 33.78
 -31.03
 1.9
 0
 0
 0
 1.49
 0.03
 2.368
 '5807003'
64
 33.76
 -24.29
 3.39
 0
 0
 0
 0.37
 0.04
 2.433
 '5101823'
65
 33.74
 -28.76
 1.86
 0
 0
 0
 0.3
 0.01
 2.93
 '5727879'
66
 33.7
 -30.61
 0.9
 0.88
 0
 0
 1.18
 0.13
 4.048
 '7988034'
67
 33.68
 -25.82
 3.22
 0
 0
 0
 1.05
 0.02
 3.306
 '7759524'
68
 33.65
 -34.64
 0.54
 0
 0
 0
 1.42
 0.25
 3.934
 '7474459'
69
 33.56
 -28.03
 2.63
 0
 0
 1.91
 0.41
 0.36
 2.679
 '6215486'
70
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 0
 0.82
 0.02
 2.14
 '7674411'
235
 26.04
 -21.24
 1.6
 0
 0
 0
 0.02
 0.03
 1.902
 '5225638'
236
 25.68
 -23.22
 0.93
 0
 0
 0
 0.17
 0.01
 1.426
 '5314675'
237
 25.58
 -25.04
 0.5
 0
 0
 0
 0.49
 0.04
 3.527
 '7630579'
238
 25.36
 -22.71
 1
 0
 0
 0
 0.17
 0
 1.366
 '5939731'
239
 25.31
 -21.11
 1.7
 0
 0
 0
 0.45
 0.01
 2.27
 '7745425'
240
 25.3
 -22.48
 1
 0
 0
 0
 0.26
 0.24
 2.717
 '7854051'
241
 25.26
 -24.16
 0.37
 0
 0
 0
 0
 0
 1.06
 '6755121'
242
 25.22
 -22.21
 1.58
 0
 0
 0
 0.89
 0.05
 2.681
 '5252659'
243
 25.03
 -24.42
 0.62
 0
 0
 0
 0.68
 0.07
 3.441
 '7690200'
244
 24.83
 -19.23
 2
 0
 0
 0
 0.2
 0
 2.244
 '9018640'
245
 24.78
 -27.29
 0.03
 0
 0
 0
 2.07
 1.54
 2.538
 '7795974'
246
 24.74
 -19.84
 1.74
 0
 0
 0
 0.15
 0
 1.369
 '5614186'
247
 24.66
 -22.01
 0.94
 0
 0
 0
 0.1
 0.02
 0.521
 '7934371'
248
 24.58
 -25.98
 0
 0
 0
 0
 0.71
 0.01
 2.055
 '7777514'
249
 24.52
 -21.72
 1
 0
 0
 0
 0.12
 0.05
 2.724
 '7733216'
250
 24.48
 -25.57
 0
 0
 0
 0
 0.7
 0.02
 2.933
 '7600192'
251
 24.43
 -24.78
 0
 0
 0
 0
 0.19
 0.03
 2.101
 '5325654'
252
 24.4
 -14.3
 3.88
 0
 0
 0
 1.17
 0.79
 0.668
 '9031574'
253
 24.37
 -18.8
 2
 0
 0
 0
 0.22
 0
 1.232
 '5666147'
254
 24.35
 -20.19
 1.56
 0
 0
 0
 0.27
 0.02
 2.359
 '5253555'
255
 24.26
 -25.8
 0.64
 0
 0
 1.35
 2
 1.88
 3.169
 '7495305'
256
 24.22
 -22.84
 0.62
 0
 0
 0
 0.51
 0.53
 1.215
 '7662712'
257
 24.2
 -16.41
 3.12
 0
 0
 0
 2.38
 3.18
 1.858
 '5319694'
258
 24.18
 -19.58
 1.63
 0
 0
 0
 0.16
 0.02
 2.095
 '6045092'
259
 24.17
 -15.69
 2.85
 0
 0
 0
 0.07
 0.05
 0.52
 '5175423'
260
 24.07
 -21.74
 1
 0
 0
 0
 0.33
 0.01
 1.034
 '5728158'
261
 24
 -24.31
 0.19
 0
 0
 0
 0.45
 0.01
 2.619
 '5131604'
262
 23.95
 -25.28
 0
 0
 0
 0
 0.76
 0.18
 0.978
 '7637949'
263
 23.92
 -18.82
 2
 0
 0
 0
 0.62
 0.33
 1.327
 '5225540'
264
 23.74
 -23.98
 0.8
 0
 0
 0.59
 1.1
 0.04
 2.987
 '7952016'
265
 23.6
 -22.76
 0.58
 0
 0
 0
 1.51
 2.14
 2.229
 '7955386'
266
 23.59
 -20.49
 1.5
 0
 0
 0
 0.72
 0.02
 2.13
 '5737089'
267
 23.57
 -15.05
 2.98
 0
 0
 0
 0.23
 0.06
 4.215
 '7984791'
268
 23.43
 -23.47
 0
 0
 0
 0
 0.02
 0.01
 3.118
 '5304628'
269
 23.24
 -21.42
 0.64
 0
 0
 0
 0.05
 0
 0.531
 '4022614'
270
 23.23
 -15.13
 2.8
 0
 0
 0
 0.15
 0
 1.656
 '7960310'
271
 23.18
 -17.22
 2
 0
 0
 0
 0.04
 0.04
 0.652
 '5309703'
272
 23.06
 -20.06
 1
 0
 0
 0
 0
 0
 1.18
 '5102936'
273
 22.95
 -20.61
 0.98
 0
 0
 0
 0.29
 0
 1.882
 '9016814'
274
 22.92
 -15.69
 2.58
 0
 0
 0
 0.25
 0
 2.203
 '9031350'
275
 22.91
 -22.97
 0
 0
 0
 0.21
 0.04
 0.23
 2.189
 '7664184'
276
 22.66
 -19.66
 1
 0
 0
 0
 0
 0
 0.813
 '5351178'
277
 22.65
 -12.49
 3.72
 0
 0
 0.1
 0.87
 0.8
 2.554
 '5930362'
278
 22.43
 -17.47
 1.82
 0
 0
 0
 0.87
 1.13
 2.362
 '5235377'
279
 22.38
 -22.58
 0
 0
 0
 0
 0.1
 0
 1.968
 '5635486'
280
 22.28
 -14.32
 2.7
 0
 0
 0
 0.08
 0
 1.308
 '5113883'
281
 22.27
 -19.32
 1
 0
 0
 0
 0.48
 0.91
 1.119
 '5403251'
282
 22.14
 -17.55
 1.93
 0
 0
 0
 0.69
 0.2
 2.096
 '5481947'
283
 22.05
 -20.89
 0.69
 0
 0
 0
 0.46
 0.01
 2.017
 '5880585'
284
 21.98
 -15.88
 2.5
 0
 0
 0
 0.71
 0.02
 0.615
 '9040725'
285
 21.95
 -11.52
 3.97
 0
 0
 0
 0.78
 0.03
 2.634
 '7774207'
286
 21.8
 -17.84
 1.96
 0
 0
 0
 1.78
 1.63
 2.403
 '7743596'
287
 21.69
 -18.71
 1
 0
 0
 0
 0.01
 0
 1.463
 '9032389'
288
 21.59
 -19.15
 0.99
 0
 0
 0
 0.27
 0
 1.085
 '7664088'
289
 21.37
 -15.61
 2
 0
 0
 0
 0.12
 0
 1.926
 '4000001'
290
 21.36
 -20.91
 1
 0.04
 0
 0
 1.35
 0.01
 1.908
 '5865114'
291
 21.23
 -13.63
 3.34
 0
 0
 0
 1.23
 0.05
 0.577
 '5109432'
292
 21.03
 -20.7
 0.88
 0
 0
 0.51
 1.97
 2.14
 3.249
 '5145823'
293
 20.99
 -17.97
 1.02
 0
 0
 0
 0.02
 0
 2.099
 '5305260'
294
 20.84
 -16.48
 1.46
 0
 0
 0
 0.23
 0.45
 1.783
 '7549075'
295
 19.82
 -16.71
 1.23
 0
 0
 0
 0.29
 0
 0.604
 '9035977'
296
 19.8
 -15.97
 1.62
 0
 0
 0
 0.6
 0.14
 3.032
 '5303377'
297
 19.5
 -14.25
 2.58
 0
 0
 0
 1.24
 0
 1.166
 '9033391'
298
 19.36
 -11.22
 3
 0
 0
 0
 0.48
 0.09
 2.85
 '9018385'
299
 19.35
 -10.95
 3
 0
 0
 0
 0.3
 0.01
 0.996
 '4022605'
300
 19.25
 -15.48
 1.3
 0
 0
 0
 0.21
 0.29
 0.36
 '9040279'
301
 19.17
 -8.75
 3.55
 0
 0
 0
 0.12
 0.01
 0.449
 '4004234'
302
 18.68
 -12.92
 1.99
 0
 0
 0
 0.11
 0
 1.23
 '5661991'
303
 17.78
 -12.97
 1.72
 0
 0
 0
 0.4
 0.46
 1.233
 '7633686'
304
 16.69
 -13.95
 1
 0
 0
 0
 0.13
 0.01
 1.394
 '9028071'
305
 16.04
 -10.11
 1.99
 0
 0
 0
 1.51
 2.97
 0.739
 '9016568'
306
 12.47
 -10.78
 0.6
 0
 0
 0
 0.05
 0.01
 0.583
 '9014885'

10/28/13: 3Q6X Docking with CB306 in GOLD
Table 2: 3Q6Xdocked to CB306 in GOLD.

#
 Score
 S(PLP)
 S(hbond)
 S(cho)
 S(metal)
 DE(clash)
 DE(tors)
 intcor
 time
 name
1
 97.74
 -41.74
 2.18
 0
 8.69
 0
 1.35
 0.01
 27.569
 '5175515'
2
 95.79
 -53.1
 0.62
 0
 7.34
 0
 1.76
 0.31
 28.672
 '7726180'
3
 95.13
 -55.98
 0
 0
 6.82
 0
 2.03
 2.19
 58.504
 '5531766'
4
 94.32
 -53.57
 0
 0
 6.96
 0
 0.54
 0.06
 38.006
 '5282931'
5
 93.19
 -40.31
 1.96
 0
 7.99
 0
 0.53
 0.11
 56.751
 '5192853'
6
 91.99
 -76.57
 0.6
 0.96
 2
 0
 0.72
 0.11
 35.223
 '9027311'
7
 91.84
 -51.6
 0
 0
 6.88
 0
 0.57
 0.08
 39.87
 '7575772'
8
 89.58
 -60.17
 2.11
 0
 3.98
 0
 0.44
 0.03
 74.036
 '7746693'
9
 87.9
 -39.14
 1
 0
 7.99
 0.98
 0.63
 0.06
 61.183
 '5467784'
10
 87.48
 -43.36
 0
 0
 7.61
 0
 1.87
 2.22
 15.206
 '6629394'
11
 87.03
 -44.38
 0.9
 0
 6.94
 0
 1.88
 2.09
 59.386
 '7676009'
12
 86.37
 -43.54
 0.84
 0
 6.93
 0
 0.65
 0.02
 10.388
 '5101823'
13
 85.35
 -41.94
 1.04
 0
 6.97
 0
 0.8
 0.06
 49.079
 '6407567'
14
 84.7
 -85.67
 0
 0
 0
 0
 0.8
 0.62
 83.242
 '7558664'
15
 83.42
 -53.2
 3.13
 0
 3.5
 0
 1.16
 2.11
 16.979
 '7959170'
16
 83.36
 -44.51
 1.15
 0
 5.99
 0
 0.3
 0.07
 16.898
 '7842136'
17
 82.74
 -44.57
 0.9
 0
 5.96
 0
 0.16
 0.01
 14.492
 '6431688'
18
 82.62
 -47.77
 0
 0
 5.99
 0
 0.57
 0.04
 34.819
 '6565654'
19
 81.55
 -56.82
 2.99
 0
 2.97
 0
 2.19
 2.22
 40.33
 '5739880'
20
 81.39
 -43.15
 3.51
 0
 4.75
 0
 1.44
 2.08
 19.797
 '7825083'
21
 81.34
 -63.98
 0.34
 0
 2.83
 0
 0.37
 0.09
 55.977
 '7759524'
22
 81.31
 -35.27
 0.83
 0
 7.28
 0
 0.08
 0.01
 7.771
 '9040855'
23
 80.96
 -51.71
 2
 0
 3.94
 0
 0.23
 0.07
 30.387
 '7724431'
24
 80.51
 -68.93
 0.93
 0
 1.96
 0
 1.5
 0.05
 65.244
 '7938694'
25
 79.53
 -37.97
 0
 0
 6.99
 0
 0.2
 0.02
 11.135
 '5135469'
26
 79.32
 -48.79
 3.09
 0
 3.58
 0
 1.17
 2.11
 17.292
 '7964554'
27
 79.31
 -37.43
 0.94
 0
 6.88
 1.6
 0.32
 0
 39.554
 '7814264'
28
 79.27
 -56.8
 0
 0
 3.83
 0
 1.79
 3.04
 18.414
 '7623092'
29
 79.14
 -50.69
 1.79
 0
 3.99
 0
 0.44
 0.05
 14.154
 '7589277'
30
 79.04
 -35.68
 0.94
 0
 6.99
 0
 0.71
 0.01
 24.478
 '6433056'















































































Week of 10/20/13 - 10/26/13
external image 4Yy2w.png
external image 4Yy4a.png

external image 4Yy5w.png

external image 4Yy6f.png

Analysis:
The activity assays don't indicate a distinct correlation between velocity and concentration such that the Km value can be determined. most of the results indicate sporadic values that are not suggestive of the trend expected. The results should indicate an increase in velocity as Nitrocefin concentration increases. initially,k we thought that the difficulty in determining the Km value came from the linear region of the Absorbance vs Time graph being too short and thus making it difficult to determine an accurate velocity. Thus, we reduced the Nitrocefin concentrations and tried the assay over a larger range of concentrations in our 2nd attempt at the activity assay. We also conducted the experiment with n=3 trials during the experiment in order to show reproducibility. However, the results of the experiment still didn't indicate the data indicative of a curve that could denote km. In order to try to increase the length of the linear region of the Absorbance vs Time curve even further such that it would be easier to determine the velocity per concentration of Nitrocefin, we reduced the enzyme concentration by 1/2, from 2ug to 1ug per 500uL of total reaction mix in the cuvette. The results still indicated poor Km relation and poor velocity vs concentration correlation.
Week of 10/13/13 - 10/19/13
Nice work Brendan. Good captions and analysis. Thank you. -Max 10/21/13
10/18/13: Enzyme Assay

external image 4UddC.png

external image 4Udhh.png

Analysis: Reduced the concentration of working dilution protein and increased the volume. Also halved the concentration
of protein in order to increase the length of the linear region of the curve. However, the data still shows sporadic data that
indicates no distinct trend. More assays to come!

10/14/13: Enzyme Assay




external image 4UcCx.png

external image 4UcDn.png
Analysis: Better results, but using GraphPad Prism (results to be shown next week due to lack of access), the Km values
were determined to be nonsense (>118). It is suspected that inconsistencies in stirring the protein into the nitrocefin could
be the cause of the problem. In order to reduce this issue, another activity assay will be conducted that uses a
larger volume of a more dilute working dilution of NDM-1 protein, so it hopefully disperses into the rest of the solution more
evenly.

Week of 10/6/13-10/12/13:

10/7/13: Enzyme assay
external image 4NWBe.jpg
external image 4NWFM.png
Thoughts on Enzyme Assay:
Experiment needs to be redone. The values received from the 20uM and 38.5uM nitrocefin assays are as expected, namely, that the
slope or velocity of the reaction increased for the greater concentration of substrate. However, this trend seemingly discontinued at the
higher concentrations of 5.56uM and 100uM nitrocefin.


10/7/13 - Control Ligand Docking in ICM Conducted
NAME
 Score
 Natom
 Nflex
 Hbond
 Hphob
 VwInt
 Eintl
 Dsolv
 SolEl
 mfScore
 POS
 RecConf
5742832aztreonampos4
 33.972006
 45
 7
 -4.279057
 -3.652
 -24.2
 0.325956
 18.418354
 80.434619
 -80.5391
 14
 1
2244asprin
 30.948482
 21
 1
 -2.977612
 -3.325
 -11.9
 0
 9.203528
 68.359647
 -75.1042
 10
 1
65894faropenempos7
 27.766007
 34
 4
 -1.899556
 -4.221
 -18.8
 0
 12.940278
 63.800214
 -103.736
 4
 1
5280980clavulanicpos5
 20.04634
 23
 4
 -1.220113
 -3.187
 -14.1
 0
 10.577421
 44.021473
 -65.5747
 15
 1
5904penicillingpos10
 14.989494
 41
 5
 -8.425242
 -4.076
 -22.9
 0.082477
 14.850178
 72.325838
 -113.689
 1
 1
5762077neg1
 14.030338
 40
 2
 -4.134254
 -5.283
 -29.9
 1.616009
 12.804248
 68.938438
 -74.9564
 8
 1
1033244neg3
 11.40441
 52
 4
 -1.032907
 -7.052
 -31.4
 0
 7.872336
 63.349512
 -61.8169
 6
 1
17399864neg4
 10.794869
 37
 2
 0
 -5.827
 -22.7
 1.968481
 8.710066
 41.929861
 -60.2192
 9
 1
20824carbenicillin
 10.145432
 44
 7
 -4.383559
 -4.716
 -22
 1.036014
 16.204445
 43.900916
 -89.0735
 12
 1
71339biapenempos6
 9.824641
 42
 4
 -1.211114
 -4.986
 -18.5
 0
 13.946948
 33.116053
 -105.377
 5
 1
33255Cefazolinpos8
 9.325897
 43
 6
 -9.472784
 -5.12
 -31
 0.518731
 24.383932
 68.627518
 -126.68
 3
 1
6436055cefcapenepos3
 9.280157
 49
 5
 -7.642363
 -4.923
 -33.8
 0
 21.734659
 70.289256
 -114.487
 13
 1
6453491ampicillinoicacidpos1
 7.550721
 46
 9
 -8.235407
 -4.123
 -21.8
 1.027698
 19.700354
 48.151559
 -72.9888
 11
 1
6249ampicillinpos9
 4.978397
 43
 6
 -8.404371
 -4.396
 -21.9
 0.99322
 15.235351
 53.13562
 -80.6112
 2
 1
3129153neg2
 4.7057
 45
 3
 -3.278469
 -5.908
 -30.8
 0.593811
 13.714974
 53.132364
 -112.503
 7
 1
Figure 1: Control ligand docking to NDM-1 betalactamase conducted in ICM. Results are strange because some of the negative
controls outranked some of the most notable positive controls such as ampicillin.

Analysis:
Need to take another look at the reasons control docking of these ligands occurs strangely. The top few ligands make sense in the
sense that aztreonam and clasvulanic acid are the only two drugs known to possibly inhibit NDM-1 betalactamse. However,
it is not yet clear why these negative controls outranked ligands such as ampicillin, because NDM-1 betalactamse cleaves
the beta-lactam ring of ampicillin.

Week of 9/30/13-10/5/13:
    • Good captions but try and add an analysis. Thank you. -Max 10/07/2013

10/5/13: ChemBridge docking to 4EY2 begun.
#
 Score
 S(PLP)
 S(hbond)
 S(cho)
 S(metal)
 DE(clash)
 DE(tors)
 intcor
 time
 Ligand name
1
 92.78
 -57.09
 3.26
 1
 3.96
 0
 1.29
 1.59
 26.476
 '33255Cefazolinpos8'
2
 91.96
 -74.85
 0
 0
 2.98
 0
 0.41
 0.04
 26.104
 '3129153neg2'
3
 91.8
 -74.59
 0
 0
 2.99
 0
 0.41
 0.03
 46.328
 '3129153neg2'
4
 91.26
 -55.82
 3.18
 1
 3.94
 0
 1.22
 1.65
 26.749
 '33255Cefazolinpos8'
5
 90.81
 -71.61
 0
 0.94
 3
 0
 0.84
 0.06
 27.16
 '3129153neg2'
6
 90.41
 -70.42
 0
 1
 2.97
 0
 0.45
 0.04
 21.168
 '3129153neg2'
7
 89.98
 -70.67
 0
 0.54
 3
 0
 0.16
 0.04
 19.195
 '3129153neg2'
8
 89.78
 -51.55
 1.36
 0
 5.85
 0
 0.64
 0.3
 27.678
 '6453491ampicillinoicacidpos1'
9
 89.08
 -72.89
 0
 0
 2.93
 0
 0.73
 0.08
 18.626
 '3129153neg2'
10
 88.6
 -52.71
 1
 0
 5.97
 0
 1.66
 0.38
 28.928
 '6453491ampicillinoicacidpos1'
11
 86.86
 -46.4
 0.52
 0
 6.95
 0
 1.65
 0.52
 13.904
 '65894faropenempos7'
12
 86.73
 -46.1
 0.63
 0
 6.91
 0
 1.61
 0.48
 13.571
 '65894faropenempos7'
13
 86.62
 -46.13
 0.8
 0
 6.84
 0
 1.72
 0.5
 14.763
 '65894faropenempos7'
14
 83.74
 -44.1
 1.57
 0
 5.96
 0
 0.66
 0.49
 61.672
 '20824carbenicillin'
15
 83.36
 -46.09
 0.54
 0
 5.99
 0
 0.33
 0.38
 62.947
 '6249ampicillinpos9'
16
 80.1
 -46.93
 3.8
 1
 3.83
 0
 3.31
 2.44
 96.139
 '6436055cefcapenepos3'
17
 80.03
 -47.92
 3.12
 1
 3.96
 0
 3.39
 2.74
 99.119
 '6436055cefcapenepos3'
18
 79.4
 -79.92
 0
 0
 0
 0.25
 2.22
 4.15
 46.051
 '1033244neg3'
19
 79.2
 -49.01
 2
 1
 3.97
 0
 1.43
 0.22
 19.093
 '71339biapenempos6'
20
 72.12
 -30.39
 0.86
 0
 6.97
 0
 1.34
 0.01
 11.773
 '2244asprin'
21
 71.95
 -33.5
 0.86
 0
 5.99
 0
 0.09
 0.09
 10.979
 '5280980clavulanicpos5'
22
 70.09
 -46.1
 4.42
 0
 1.97
 0
 2.45
 3.81
 64.004
 '5742832aztreonampos4'
23
 69.94
 -64.97
 0
 0
 0.97
 0
 0.42
 0.01
 13.793
 '5762077neg1'
24
 68.43
 -71.65
 0
 0
 0
 2.89
 0.36
 0.38
 31.331
 '17399864neg4'
25
 67.4
 -51.31
 1.84
 0
 1.96
 0
 0.72
 0.25
 19.105
 '5904penicillingpos10'
26
 61.51
 -58.98
 1
 0
 0
 0
 0.24
 0
 9.694
 '17399864neg4'
Figure 1: Control Docking results from GOLD. 26 ligand-prepped results shown. One of the negative controls
scored very highly. The Autoscale was set to 2.


10/4/13: Control Docking completed. Begin ligand prep on the ChemBridge Library.
10/1/13: Ligand Control prep completed for virtual
9/31/13: Protein Prep completed for Virtual



Week of 9/23/13-9/29/13:
9/26/13: FPLC

nanodrop_092613_NDM1.jpg
Figure 1: Nanodrop of Elution 1 of NDM-1 protein after Ni-NTA purification, prior to FPLC protocol.


20130926BrendanChou_FPLCresults_NDM1.jpg
Figure 2: FPLC protocol graphical analysis. Tubes 39-48 taken for reconcentration.

bc24675-2.JPG

Figure 3: Nanodrop results of NDM-1 Elution 1 after FPLC purification.


Week 3 & 4 9-Sep - 22-Sep
Brendan - good work. Move forward to enzyme assays. Dr. B 100113
PS - interesting grid on your master plate...?


Week of 9/16/13-9/22/13:

September 19, 2013: Continue washing off excess dye. Need to FPLC due to the presence of other bands.
weeee.JPG
P7711_thumb
P7711_thumb

Figure 1: Gel of protein NDM-Beta-lactamase after purification. Lanes from left to right: Ladder,
Flow through, Wash, Elution 1, Elution 2, Soluble Fraction. The ColorPlus protein ladder is
shown to the below with the different MWs shown beside each band.

Gel looks pretty successful. the band is darkest for the Elution 1 sample and is considerably lighter
for the Elution 2 sample. There are still significant bands at other MW, so FPLC will be performed
for further purification starting next week.

The protein target has a molecular weight of 28kDa. This seems to be where the ladder indicates the
darkest band is showing up in the Elution samples.

September 18, 2013: Ran gel of protein. Dyed and now washing off excess dye. Tomorrow = imaging.
September 16, 2013: Purification and storage. Next up: characterization.




Week of 9/9/13-9/15/13
September 13, 2013: Lysing of cells occurred. After lysing, stored in fridge after spin down and initial filtration to await next week's purification

September 12, 2013: Centrifuge down of the cells at 4 degrees C and 6300 rpm. Glycerol stocks made and placed in cryovials in the -80C fridge.
Tube 1: 3.15g Tube 2: 1.98g

September 11, 2013: Relatively few cultures of bacteria seen, this is expected. Started culturing after OD600 of ~0.5 was reached. Kan was added late, and OD600 was closer to 0.8 than 0.5 when placed in a room temperature shaker for overnight. Glycerol Stocks protocol Day 1 started. Transfer to cryovials tomorrow.

2013-09-11 15.39.34.jpg

Figure 1: Plated B21(DE3) E.Coli transformed with p27bNDM(35a) on LB+Agar+Sucrose
plates. 50uL of culture added to plate.

2013-09-11 15.39.40.jpg

Figure 2: Plated B21(DE3) E.Coli transformed with p27bNDM(35a) on LB+Agar+Sucrose
plates. 10uL of culture added to plate.

September 10, 2013: Only a few colonies remaining. Perhaps 30uL/mg as indicated by Fast lab protocol should be taken into advisement. However, this could also have been a product of the timing after heat shocking. Made two culture flasks, one with 30uL/mL Kan, and the other with 50uL/mL Kan in case the 50uL/mL Kan culture doesn't turn out turbid.
September 9, 2013: Transformation on to Kan plates. Made fresh LB for the next day's culturing. Note: Didn't allow for full 2min after heat-shocking before adding SOC media.


Week of 9/2/13-9/8/13
Good - Dr. B 090913

September 6, 2013:
Target Group Meeting.
To-Do List:
1. Wet Lab
-Transformation+Expression [https://docs.google.com/file/d/0B_Gl3lMyhDsoSWhzZ2FQaXk1dk0/edit?usp=sharing]


-9/16 -Purification <2hrs-2.5hrs> [https://docs.google.com/file/d/0B_Gl3lMyhDsoQzk2SUJ5TDYzQUk/edit?usp=sharing] [store in 4C]
-9/18 -Characterization <3hrs> [https://docs.google.com/file/d/0B_Gl3lMyhDsoQ2k1czMxM240dzg/edit?usp=sharing]
-<Serena> Preliminary assay - Nitrocefin at varying concentrations

2. Virtual - with the aid of the gracious Madeline :D
-GOLD screen preliminary library:


-ICM/VINA Docking, ICM Screen certain attributes and compare results to GOLD ligand docking vs attribute-filtered library
-GraphPad Prism - enzyme kinetics determination


September 4, 2013:
Results from DNA-Sequencing received and analyzed.
Blastn searches run for both of these sequences.

T7:

NNNNNNNNNNNNCNTCTAGANTAATTTTNTTTAACTTTAAGAAGGAGATATACATATGAAATACCTGCTGCCGACCGCTG
CTGCTGGTCTGCTGCTCCTCGCTGCCCAGCCGGCGATGGCCATGGGCCAGCAAATGGAAACTGGCGACCAACGGTTTGGC
GATCTGGTTTTCCGCCAGCTCGCACCGAATGTCTGGCAGCACACTTCCTATCTCGACATGCCGGGTTTCGGGGCAGTCGC
TTCCAACGGTTTGATCGTCAGGGATGGCGGCCGCGTGCTGGTGGTCGATACCGCCTGGACCGATGACCAGACCGCCCAGA
TCCTCAACTGGATCAAGCAGGAGATCAACCTGCCGGTCGCGCTGGCGGTGGTGACTCACGCGCATCAGGACAAGATGGGC
GGTATGGACGCGCTGCATGCGGCGGGGATTGCGACTTATGCCAATGCGTTGTCGAACCAGCTTGCCCCGCAAGAGGGGAT
GGTTGCGGCGCAACACAGCCTGACTTTCGCCGCCAATGGCTGGGTCGAACCAGCAACCGCGCCCAACTTTGGCCCGCTCA
AGGTATTTTACCCCGGCCCCGGCCACACCAGTGACAATATCACCGTTGGGATCGACGGCACCGACATCGCTTTTGGTGGC
TGCCTGATCAAGGACAGCAAGGCCAAGTCGCTCGGCAATCTCGGTGATGCCGACACTGAGCACTACGCCGCGTCAGCGCG
CGCGTTTGGTGCGGCGTTCCCCAAGGCCAGCATGATCGTGATGAGCCATTCCGCCCCCGATAGCCGCGCCGCAATCACTC
ATACGGCCCGCATGGCCGACAAGCTGCGCGCTAGCCAGCCAGAACTCGCCCCGGAAGACCCCGAGGATGTCGAGCACCAC
CACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGANTTGGCTGCTGCCACCGCTGANCAATAACT
AGCATAACCCCTTGGGGNNCTAAACGGGTCTTGNNGGGTTTTTGCTGAAGGNNNNTATATCCGNATTGGCGAATGGGNNG
NNNCNGNAGNNNGCATTNAGNNNGNNNNNNGNNNNCNCNCANCNNNACNCTANNCTTNCNGCNNNANNNCCNNNNNTTCN
NTTTCNTNNNNNTNNNNNNCNNNNNTNNNNNNTTTNCNNNANNNNNAANNGGGGNNCNCNNNNNGNNNNNNTNNNNNNNN
GNNNNCNNNNNNNNANANNTNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNN
Figure 1: DNA-Sequencing results from CORE after Midi-prep of pET-27b(+) NDM-1(d35) plasmid. Shown with theT7 in-house primer.
T7Term

NNNNNNNNNNNNNNNTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGACATCCTCGGGGTCT
TCCGGGGCGAGTTCTGGCTGGCTAGCGCGCAGCTTGTCGGCCATGCGGGCCGTATGAGTGATTGCGGCGCGGCTATCGGG
GGCGGAATGGCTCATCACGATCATGCTGGCCTTGGGGAACGCCGCACCAAACGCGCGCGCTGACGCGGCGTAGTGCTCAG
TGTCGGCATCACCGAGATTGCCGAGCGACTTGGCCTTGCTGTCCTTGATCAGGCAGCCACCAAAAGCGATGTCGGTGCCG
TCGATCCCAACGGTGATATTGTCACTGGTGTGGCCGGGGCCGGGGTAAAATACCTTGAGCGGGCCAAAGTTGGGCGCGGT
TGCTGGTTCGACCCAGCCATTGGCGGCGAAAGTCAGGCTGTGTTGCGCCGCAACCATCCCCTCTTGCGGGGCAAGCTGGT
TCGACAACGCATTGGCATAAGTCGCAATCCCCGCCGCATGCAGCGCGTCCATACCGCCCATCTTGTCCTGATGCGCGTGA
GTCACCACCGCCAGCGCGACCGGCAGGTTGATCTCCTGCTTGATCCAGTTGAGGATCTGGGCGGTCTGGTCATCGGTCCA
GGCGGTATCGACCACCAGCACGCGGCCGCCATCCCTGACGATCAAACCGTTGGAAGCGACTGCCCCGAAACCCGGCATGT
CGAGATAGGAAGTGTGCTGCCAGACATTCGGTGCGAGCTGGCGGAAAACCAGATCGCCAAACCGTTGGTCGCCAGTTTCC
ATTTGCTGGCCCATGGCCATCGCCGGCTGGGCAGCGAGGAGCAGCAGANCAGCAGCAGCGGTCGGCAGCAGGTATTTCAT
ATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGNTATCCGCTCACAATTCCCCTATAGTGAG
TCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACNCCGNANGCATCGNGGNNGGCATCACCGGNNCCNNNGNTGC
NGNTGCTGGCGNCTATATCNNNANNTCNNCNAATGGGGNNNNTNGGGCNCGCCNCTTNGNCTCNTGNANNNNTGTTCNGN
NNNNGGGNNNGGNNGNNGNCCCNNNNNNNNNNTNNNNGNNNCNTCNCNTNNNNNNNNNCNNNNNNNCGNNGGNGGNNNNN
ANNGNNNNCNNCNNNNNGNNNNNNTTNNANNNANNANTNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNNNNNGN
Figure 2: DNA-Sequencing results from CORE after Midi-prep of pET-27b(+) NDM-1(d35) plasmid. Shown with theT7term in-house primer.



external image 4jlYl.png

Figure 3: Blastn search results of the DNA sequence received from the CORE. Blastn search (of T7Term primer+plasmid)
confirms the presence of the pET-27b(+) NDM-1(d35) plasmid.


external image 4jme2.png

Figure 4: Blastn search results of the DNA sequence received from the CORE. Blastn search (of T7 primer+plasmid)
confirms the presence of the pET-27b(+) NDM-1(d35) plasmid.

September 2, 2013:
Midi prep of DH5a transformed with pET-27b(+) NDM-1(d35). And nanodrop of results.
Plasmids sent for sequencing using the T7term and T7 in-house primers provided by the CORE.



midiprep results_wedabest.JPG
Figure 1: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are too low, and the graph indicates a negative 230 value.

midiprep results_wedabest_2.JPG
Figure 2: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are too high, and the graph indicates a negative
230 value.

midiprep results_wedabest_3.JPG

Figure 3: Nanodrop of p276-NDM(35) after Midi-prep. 260/230
values are now as Expected. Used new blanking TE Buffer and
new nanopure water for blanking and calibrating.

midiprep results_wedabest_4.JPG

Figure 4: Nanodrop of p276-NDM(35) after Midi-prep.
260/230 values are now as expected. Used new blanking
TE Buffer and new nanopure water for blanking and calibrating.



Week of 8/28/13 - 9/1/13:

August 31, 2013: Spin down and fridge colonies after 20hrs.
August 30, 2013: Took two colonies and incubated overnight after autoclaving LB media.
August 29, 2013: Transformed DH5a with pET27b(+) NDM-1(d35) on Kan plates, for later Midi-prep. Running low on plasmid.



Week 3:


Protein expression completed.

external image 3nfiS.png

Figure 1: Blast results of GFP plasmid sent for sequencing.

6.17.13 GFP Gel Electrophoresis.png

Figure 2: Gel electrophoresis of GFP DNA post-PCR. Lane 1 and 6 = Ladder, Lane 2 and 7 = 1ng GFP,
Lane 3 and 8 = 5ng GFP, Lane 4 and 9 = 25ng GFP. Lane 5 and 10 = 0ng. Lanes 1-5 with PCR with M13
primers, at M13 primer optimal temperatures, and Lanes 6-10 with PCR with VDS1 and VDS2 primers, at
M13 primer optimal temperatures.



Week 2:

Trial 1.jpg

Figure 1: Nanodrop measurement of pNIC-Bsa4 after PCR. Concentration of 301.3 ng/uL exhibited. A sample of this plasmid solution
was sent to sequencing to confirm Midi-prep results.

trial 2.jpg

Figure 2: Nanodrop measurement of pNIC-Bsa4 after PCR. Concentration of 283.0 ng/uL exhibited. A sample of this plasmid solution
was sent to sequencing to confirm Midi-Prep results.

6.13.13 Pgbr22 Gel Electrophoresis.png
Figure 3: Brendan (Right) pgbr22 post-PCR. About 1kb.
Lane 6 = Ladder, Lane 7 = 1ng pgbr22, Lane 8 = 5ng pgbr22,
Lane 9 = 25ng pgbr22. Lane 10 = 0ng Lanes 1-5=Nicolet’s.


Week 1
6.6.13_JPB_PNIC_Bsa4_culture_A_1ng_plasmid.jpeg
Figure 1: E. coli cells transformed with 1 ng pNIC-Bsa4 plasmid grown on LB+Amp Agar plates.
Approximately 1648 colonies/ng plasmid.

6.6.13_JPB_PNIC_Bsa4_culture_B_5ng_plasmid_Top_view.jpeg
Figure 2: E. coli cells transformed with 5 ng pNIC-Bsa4 plasmid grown on LB+Amp Agar plates.
Approximately 3072 colonies/ng plasmid.


6.6.13_JPB_PNIC_Bsa4_culture_C_25ng_plasmid.jpeg
Figure 3: E. coli cells transformed with 25 ng pNIC-Bsa4 plasmid grown on LB+Amp Agar plates.
Approximately 689 colonies/25ng, = 27.6coloines/ng plasmid.

Nanodrop A.jpeg

Nanodrop B.JPG